Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.     

FGF9 is an autocrine and paracrine prostatic growth factor expressed by prostatic stromal cells

Polypeptide growth factors, including members of the fibroblast growth factor (FGF) family, play an important role in the growth and maintenance of the normal prostate. We have found that FGF9 is expressed at high levels in the normal peripheral and transition zone of the human prostate. Analysis of FGF9 production by primary cultures of prostatic epithelial and stromal cells has shown that FGF9 is produced and secreted by the prostatic stromal cells. Neither of these processes appears to be modulated by androgens. Production of FGF9 by stromal cells in vivo was confirmed by immunohistochemistry. FGF9 is a potent mitogen for both prostatic epithelial and stromal cells in culture and is a more potent mitogen for these cells than either FGF2 or FGF7, two other FGFs expressed in the human prostate. FGF9 is an abundant secreted growth factor that can act as both a paracrine mitogen for epithelial cells and an autocrine mitogen for stromal cells. Western blot analysis of tissue extracts from the normal and hyperplastic transition zone shows that FGF9 is present at two to threefold higher levels in the hyperplastic transition zone. Overexpression of this paracrine and autocrine growth factor may play an important role in the epithelial and stromal proliferation in benign prostatic hyperplasia. J. Cell. Physiol. 180:53–60, 1999. © 1999 Wiley-Liss, Inc.     

Oxytocin,oxytocin-associated neurophysin and the oxytocin receptor in the human prostate

Oxytocin has been implicated in the regulation of prostate growth. However, the cellular localisation of oxytocin in the normal and diseased human prostate is not known. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were detected by immunohistochemistry in tissues from patients undergoing routine prostatectomy and in normal human prostate epithelial and stromal cell lines. Western blot analysis detected a single band at 14 kDa with neurophysin antiserum and a 66-kDa band with oxytocin receptor antiserum in epithelial and stromal cell lines. Similar sized bands were also detected in extracts of hyperplastic and adenocarcinomic prostate tissues. Oxytocin, oxytocin-associated neurophysin and oxytocin receptor were present in stromal and epithelial cell lines and in tissue from patients with benign prostatic hyperplasia. The peptides were localised predominantly to the epithelial cells, although discrete areas of stromal staining were also observed. There was a significant difference in the intensity of oxytocin-staining between tissue displaying benign prostatic hyperplasia and invasive carcinoma, with less immunoreactivity being present in the malignant epithelial cells. Thus, oxytocin and its neurophysin and receptor are present in epithelial and stromal cells of the human prostate. Oxytocin expression is reduced with tumour progression and may provide a marker for invasive disease.This work was supported by a Project Grant (007756) from the Wellcome Trust and from Lottery Health Research     

Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.     

Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.     

Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment

Androgens and estrogens, acting via their respective receptors, are important in benign prostatic hyperplasia (BPH). The goals of this study were to quantitatively characterize the tissue distribution and staining intensity of androgen receptor (AR) and estrogen receptor-alpha (ERα), and assess cells expressing both AR and ERα, in human BPH compared to normal prostate. A tissue microarray composed of normal prostate and BPH tissue was used and multiplexed immunohistochemistry was performed to detect AR and ERα. We used a multispectral imaging platform for automated scanning, tissue and cell segmentation and marker quantification. BPH specimens had an increased number of epithelial and stromal cells and increased percentage of epithelium. In both stroma and epithelium, the mean nuclear area was decreased in BPH relative to normal prostate. AR expression and staining intensity in epithelial and stromal cells was significantly increased in BPH compared to normal prostate. ERα expression was increased in BPH epithelium. However, stromal ERα expression and staining intensity was decreased in BPH compared to normal prostate. Double positive (AR and ERα) epithelial cells were more prevalent in BPH, and fewer double negative (AR and ERα) stromal and epithelial negative cells were observed in BPH. These data underscore the importance of tissue layer localization and expression of steroid hormone receptors in the prostate. Understanding the tissue-specific hormone action of androgens and estrogens will lead to a better understanding of mechanisms of pathogenesis in the prostate and may lead to better treatment for BPH.     

Induction of Smooth Muscle Cell Phenotype in Cultured Human Prostatic Stromal Cells

Stromal cells are key regulators of growth and differentiation in the adult human prostate. Alterations in the stroma are believed to initiate the development of benign prostatic hyperplasia, and stromal–epithelial interactions may have a role in malignant progression. The prostatic stroma is composed of two major cell types, smooth muscle cells and fibroblasts. Cell cultures from the prostatic stroma have been established by several investigators, but the phenotype of these cells has not been extensively characterized and it is not clear whether they are fibroblastic or smooth muscle-like. In this study, the response of stromal cells cultured from normal prostatic tissues to transforming growth factor-β (TGFβ) was investigated. We confirmed a previous report that TGFβ inhibited the growth of prostatic stromal cells in serum-containing medium, and showed that inhibition also occurred in serum-free medium. Growth inhibition by TGFβ was irreversible after 24 to 72 h of exposure. In the absence of TGFβ, cells were fibroblastic and expressed vimentin and fibronectin but little α-smooth muscle actin. After 3 days of exposure to 1 ng/ml of TGFβ, the majority of cells expressed α-smooth muscle actin and desmin, as demonstrated by immunocytochemistry and immunoblot analysis. This effect was specific and α-smooth muscle actin was not induced by two other growth-inhibitory factors, retinoic acid or 1,25-dihydroxyvitamin D₃. These results suggest that TGFβ is an important regulator of growth and differentiation of prostatic stromal cells and that a smooth muscle cell phenotype is promoted in the presence of TGFβ.     

Characterization of cell types in human breast tumor primary cultures

Primary cultures of cells from breast carcinomas were attempted in 74 cases. Growth was observed in 46 cases. Using immunochemical demonstration of keratin proteins (KER), epithelial membrane antigen (EMA) and carcinoembryonic antigen (CEA), three morphologically distinct cell populations were characterized and described. Two cell types (E- and E'-cells) were identified as epithelial by their positive staining for KER and EMA. The third type (F cells) displayed a negative staining for these two epithelial markers; they were considered as stromal cells (fibroblasts). More than 50% of the cultures consisted of pure epithelial cells. Positive CEA staining was observed only in KER- and EMA-positive cells. Out of the 30 cultures, 15 displayed positive staining for CEA. In 7 of these, 30-50% of cells displayed positive, diffuse staining for CEA. The other 8 cultures consisted of more than 50% CEA-positive cells. Strong and homogeneous positivity was restricted to the E-cells, while in the same cultures, E'-cells displayed heterogeneous and diffuse staining. Efficiency and value of this cell culture system are discussed, in comparison with other human breast tumor cell (HBTC) culture techniques. Identification of growing cells and cellular composition of primary cultures are emphasized.     

Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co‐culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non‐macromastic epithelial cells when co‐cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia‐derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co‐culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co‐cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy.     

Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development

This review on normal and neoplastic growth of the prostate emphasizes the importance of epithelial-mesenchymal/stromal interactions. Accordingly, during prostatic development urogenital sinus mesenchyme (a) specifies prostatic epithelial identity, (b) induces epithelial bud formation, (c) elicits prostatic bud growth and regulates ductal branching, (d) promotes differentiation of a secretory epithelium, and (e) specifies the types of secretory proteins expressed. In reciprocal fashion, prostatic epithelium induces smooth muscle differentiation in the mesenchyme. Epithelial-mesenchymal interactions during development continue postnatally into adulthood as stromal-epithelial interactions which play a homeostatic role and in so doing reciprocally maintain epithelial and stromal differentiation and growth-quiescence. Prostatic carcinogenesis involves perturbation of these reciprocal homeostatic cell-cell interactions. The central role of mesenchyme in prostatic epithelial development has been firmly established through analysis of tissue recombinants composed of androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative epithelium. These studies revealed that at the very least ductal morphogenesis, epithelial cytodifferentiation, epithelial apoptosis and epithelial proliferation are regulated by stromal and not epithelial androgen receptors. Likewise, progression from non-tumorigenesis to tumorigenesis elicited by testosterone plus estradiol proceeds via paracrine mechanisms. Thus, stromal-epithelial interactions play critical roles in the hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.     

前列腺组织中神经生长因子(NGF)表达的研究

研究前列腺组织中神经生长因子（ＮＧＦ） 的生理学意义。采用原位杂交和免疫组化法, 检测４３ 例前列腺增生组织, ８ 例腺癌组织和８ 例正常组织中β－ＮＧＦｍ ＲＮＡ及其蛋白的表达及分布。结果显示β－ＮＧＦｍ ＲＮＡ 在正常组织及增生组织中定位于间质细胞, 偶见于上皮细胞中; 而在癌组织中, 上皮细胞和间质细胞有同样强度的β－ＮＧＦｍ ＲＮＡ染色。其蛋白在良性组织中表达主要着色在间质细胞中,上皮细胞呈弱表达,而癌组织中上皮细胞见着色明显增强（Ｐ＜ ０．０５）。ＮＧＦ的自分泌异常可见是前列腺组织由良性向恶性转变的原因之一。     

Defined medium for normal adult human prostatic stromal cells

Summary Stromal-epithelial interactions are pivotal in many aspects of prostatic biology. A defined culture system is critical for the investigation of factors that regulate the growth and differentiation of human prostatic stromal cells. We have identified conditions which promote stromal cell attachment and proliferation in serum-free medium. MCDB 201, originally developed for the clonal growth of chick embryo fibroblasts, proved to be a superior basal medium of those that we tested. Supplementation of MCDB 201 with basic fibroblast growth factor (FGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) permitted attachment and exponential growth of cells throughout a 7-d period with an initial inoculum as low as 10³ cells per well of a 96-well microtiter dish. Using these assay conditions, we subsequently verified that basic FGF and IGF, but not PDGF, were required for optimal growth. No activity was found for heparin, transferrin, or the androgen R1881. Epidermal growth factor (EGF) didn’t stimulate growth when added to medium containing basic FGF and IGF, but was moderately stimulatory when added to basal medium alone. Cholera toxin inhibited growth. This simple and efficient culture medium provides a suitable assay system for more extensive studies of growth regulation and differentiation of human prostatic stromal cells, and will provide the basis for future development of a defined medium that supports clonal growth. Characterization of stromal-epithelial interactions will be facilitated by the use of this defined culture system for stromal cells in conjunction with the serum-free culture systems previously developed for human prostatic epithelial cells.     

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.     

A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.     

Collagen and the proliferation and differentiation of rat ventral prostate epithelial cells

The purpose of this study was to determine if a cause-and-effect relationship exists between androgen-induced changes in collagen and epithelial cell proliferation and/or differentiation in rat ventral prostate. Analyses of the temporal relationship between dihydrotestosterone (DHT)-induced changes in the synthesis and levels of collagen in the regressed ventral prostates of adult castrates demonstrated that, during the first 7 days of restoration of prostatic growth, androgen increased the synthesis as well as the degradation of collagen. Cis-hydroxyproline (CHP) treatment (2-200 mg/kg) during the first 7 days of androgen-stimulated prostatic growth, combined with maintenance of animals on a proline-free diet, produced a dose-dependent reduction in prostate weight and DNA content to a maximum of 50%. The epithelium was characterized by numerous disorganized layers of irregularly shaped and tightly packed cells, many of which had no contact with the basal lamina. There was a loss of epithelial lamina lucida and the development of a ragged lamina densa. Cis-hydroxyproline effects were reversible in that, following cessation of CHP treatment, the perturbed morphology, DNA content, and organ weight returned to the range of DHT-treated controls. Collagenous components seem to be important in supporting the normal androgen-dependent proliferation and differentiation of prostatic epithelial cells.     

TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37°C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (≥50 μm) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusionP<0.05) and the adhesion (up to four-fold by crystal violet staining,P<0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-α or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mmn-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mm sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.     

Effect of keratinocyte growth factor on cell viability in primary cultured human prostate cancer stromal cells

In normal prostate, keratinocyte growth factor (KGF), also known as fibroblast growth factor-7 (FGF-7) serves as a paracrine growth factor synthesized in stromal cells that acts on epithelial cells through its receptor, KGFR. KGF and KGFR were found in human cancer epithelial cells as well as stromal cells. Since KGF expressed in epithelial cells of benign prostatic hyperplasia (BPH) and in prostate cancer, it has been suggested that KGF might act as an autocrine factor in BPH and prostate cancer. To investigate the roles of KGF in cancerous stroma, primary cultured human prostate cancer stromal cells (PCSCs) were isolated and evaluated. These PCSCs possessed estrogen receptors and KGFR, but not androgen receptor as determined by RT-PCR and Western blot, respectively. KGF exhibited mitogenic and anti-apoptotic effects that correlated with induction of cyclin-D1, Bcl-2, Bcl-xL and phospho-Akt expression in PCSCs, where treatment with KGF antiserum abolished cell proliferation and anti-apoptotic protein expression. PCSCs exposed to KGF for various time periods resulted in phosphorylation of Akt and subsequent up-regulation of Bcl-2. KGF modulated dynamic protein expression indicated that KGF triggered cell cycle machinery and then activated anti-apoptotic actions in PCSCs. Cell proliferation analysis indicated that tamoxifen or ICI 182,780 reduced cell viability in a dose-dependent manner; however, KGF prevented this inhibition, which further demonstrated KGF triggered anti-apoptotic machinery through activating Bcl-2 and phospho-Akt expression. In summary, KGF has an autocrine effect and serves as a survival factor in primary cultured human prostate cancer stromal cells.     

Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas

Angiogenesis sustains tumor growth and metastasis, and recent studies indicate that the vascular endothelium regulates tissue mass. In the prostate, androgens drive angiogenic inducers to stimulate growth, whereas androgen withdrawal leads to decreased vascular endothelial growth factor, vascular regression and epithelial cell apoptosis. Here, we identify the angiogenesis inhibitor pigment epithelium-derived factor (PEDF) as a key inhibitor of stromal vasculature and epithelial tissue growth in mouse prostate and pancreas. In PEDF-deficient mice, stromal vessels were increased and associated with epithelial cell hyperplasia. Androgens inhibited prostatic PEDF expression in cultured cells. In vivo, androgen ablation increased PEDF in normal rat prostates and in human cancer biopsies. Exogenous PEDF induced tumor epithelial apoptosis in vitro and limited in vivo tumor xenograft growth, triggering endothelial apoptosis. Thus, PEDF regulates normal pancreas and prostate mass. Its androgen sensitivity makes PEDF a likely contributor to the anticancer effects of androgen ablation.     

Immunohistochemical comparative analysis of transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor in normal, hyperplastic and neoplastic human prostates.

Immunoreaction to TGF-alpha was limited to the basal epithelial cells of focal areas in the normal prostates. In benign prostatic hyperplasia (BPH) the immunostained areas were more widespread and immunolabelling was observed in both basal and columnar (secretory) cells of the epithelium. Some cells in the connective tissue stroma were also stained. In prostatic adenocarcinoma, epithelial immunostaining was even more extensive and intense than in BPH, and some stromal cells were also stained. Epidermal growth factor (EGF) immunostaining was only present in some basal cells in normal prostates. In BPH, this immunoreaction was strong in the basal cells and even stronger in the secretory cells. In prostatic cancer, the intensity of epithelial cell immunoreactivity was intermediate between that of normal prostates and that of BPH specimens. EGF-receptor immunostaining was focal and located in the basal cells in normal prostates. In BPH, labelling was also localized in basal cells but extended to wider areas. Some stromal cells appeared weakly labelled. In the prostatic carcinoma, both basal and columnar cells appeared stained and the number of immunolabelled stromal cells was higher than in BPH. The results presented suggest that, in normal conditions, EGF and TGF-alpha act as autocrine growth factors for the basal cells of the prostatic epithelium. In BPH this action is maintained and, in addition, the columnar cells start to secrete both factors which are bound by the basal cell receptors, giving rise to a paracrine regulation which probably overstimulates basal cell proliferation. In prostatic carcinoma, besides these regulatory mechanisms, the acquisition of EGF-receptors by the secretory cells develops an autocrine regulation which might induce their proliferation.